Redox regulation of intestinal T cells

描述(申请人提供)：T细胞受体发出的信号转导的调节可由外源性细胞因子、细胞间接触和内源性代谢物介导，产生对抗原刺激的特殊功能反应。尽管肠道附近的免疫学环境充满敌意，但粘膜固有层T细胞(LPT)对来自“安全”管腔蛋白的抗原仍反应迟钝，但仍能对病原体产生强烈的免疫反应。因此，我们建议研究调节粘膜T细胞低反应性维持的机制，以及使这种通常静止的粘膜T细胞能够在需要时做出反应的环境信号。T细胞受体(TCR)的信号转导对细胞增殖、Fas配体、受体和黏附蛋白等免疫介质的表面表达、分化、激活诱导的细胞死亡和细胞因子基因的表达等所有功能反应都是至关重要的。在本提案中，我们将重点讨论氧不完全还原产生的活性氧物种(ROS)，如过氧化氢，它们充当第二信使，从而成为TCR发出的信号转导的内源调节器。我们最近报道，LPT对TCR交联性反应的能力减弱，通过弱增殖来衡量，反映在它们从TCR启动信号转导的能力急剧下降。为了理解LPT信号能力的下降，我们研究了直接与TCR的a链和？链相连的酶途径，重点是与TCR/CD3复合体相关的酪氨酸激酶和磷酸酶之间潜在的失衡。由于质膜很容易被过氧化氢渗透，外源低浓度、无毒的过氧化氢可以刺激酪氨酸磷酸化模式，反映TCR的参与。与观察到的TCR交联类似，过氧化氢诱导的LPT中酪氨酸磷酸化显著减弱。因此，我们假设并报道了低反应性LPT的氧化还原状态倾向于增强还原能力。此外，克罗恩病(炎症性肠病的一种)LPT对TCR结扎反应强烈(例如，增殖、干扰素？产生)，这种炎症T细胞中的氧化还原状态是氧化的，这使得TCR参与后能够进行强大的信号转导。与我们的发现一致的是，最近有报道称，在正常肠道中，固有层巨噬细胞不分泌半胱氨酸，而半胱氨酸是LPT调节其胞浆氧化还原电位所必需的。这些结果支持以下中心假设：粘膜T细胞的细胞内还原能力调节该细胞产生细胞内活性氧的能力，改变了T细胞受体在正常肠粘膜中启动免疫反应的阈值。这一假设将在以下两个具体目标中得到解决：目标1.确定TCR激活的LPT中产生的活性氧物种合成和/或稳定性的内在缺陷。目的2.建立胞浆氧化还原电位为LPT反应设定阈值。 相关性：为了保护我们免受感染，我们有一个复杂的白细胞系统，其中的主体被称为T淋巴细胞，它为宿主防御做出战略决定。从结肠或小肠分离的T细胞与从血液中分离的T细胞相比，通常反应非常弱，而从感染或肠痉挛和慢性炎症引起的肠道疾病患者的炎症肠区分离的T细胞反应又有另一个明显的特征。这一提议将定义粘膜T细胞的那些固有属性，使其能够在这些反应和不反应状态之间切换。我们最近发现，T细胞内的化学平衡(称为氧化还原电位)调节它对外部世界的反应是弱还是强。我们还表明，通过将粘膜T细胞内的平衡转换为另一种类型，这种经治疗的肠道T细胞现在的行为更像是正常的血液来源T细胞。这个项目将定义导致这种失衡的T细胞中的确切分子。然后，我们可以针对这种分子进行“纠正”，并在未来确定这种类型的治疗是否对粘膜感染、疫苗开发或粘膜自身免疫性疾病有效。

DESCRIPTION (provided by applicant): Modulation of signal transduction emanating from the T cell receptor can be mediated by exogenous cytokines, cell-cell contact, and endogenous metabolites, yielding a specialized functional response to antigenic stimulation. Despite the hostile immunological neighborhood of the intestinal tract, mucosal lamina propria T cells (LPT) remain hyporesponsive to antigens derived from "safe" luminal proteins; yet retain their capacity to mount a vigorous immune response to pathogens. We therefore propose to investigate the mechanisms regulating the maintenance of hyporesponsiveness in mucosal T cells and the environmental signals that enable this normally quiescent mucosal T cell to respond when needed. Signal transduction emanating from the T cell receptor (TCR) is critical for all functional responses, such as proliferation, surface expression of immune mediators such as Fas Ligand, receptors, and adhesion proteins, differentiation, activation induced cell death, and cytokine gene expression. In this proposal we will focus on reactive oxygen species (ROS) derived from incomplete reduction of oxygen, such as hydrogen peroxide, acting as second messengers and thus endogenous modulators of signal transduction emanating from the TCR. We recently reported that the diminished capability of LPT to respond to TCR cross-linking, as measured by weak proliferation, is mirrored by a dramatic loss in their ability to initiate signal transduction from the TCR. To understand this decrease in LPT signaling capacity we investigated the enzymatic pathway directly linked to the a and ¿ chains of the TCR, focusing on a potential imbalance between tyrosine kinases and phosphatases associated with the TCR/CD3 complex. Since the plasma membrane is readily permeable to hydrogen peroxide, exogenous administration of low, non-toxic concentrations of H2O2 stimulates a tyrosine phosphorylation pattern that reflects TCR engagement. Similar to what is observed with TCR cross-linking, H2O2-induced tyrosine phosphorylation in LPT is significantly muted. We therefore hypothesized and then reported that the redox status of the hyporesponsive LPT is tilted toward increased reductive capability. In addition, the Crohn's disease-derived (a form of inflammatory bowel disease) LPT responds robustly to TCR ligation (e.g., proliferation, IFN-? production), and the redox status in this inflamed T cell is oxidative, which enables strong signal transduction after TCR engagement. Consistent with our findings it was recently reported that in the normal intestine, lamina propria macrophages do not secrete cysteine, which is required by the LPT to regulate their cytosolic redox potential. These results support the following central hypothesis: The intracellular reducing capacity of the mucosal T cell, which modulates that cell's ability to generate intracellular reactive oxygen species, alters the threshold at which engagement of the T cell receptor initiates an immune response in the normal intestinal mucosa. This hypothesis will be addressed in the following two specific aims: Aim 1. Define the intrinsic defect in the synthesis and/or stability of reactive oxygen species generated in TCR-activated LPT. Aim 2. Establish that cytosolic redox potential sets the threshold for an LPT response. RELEVANCE: To protect us from infections we have a complex system of white blood cells, the principal of these is called the T lymphocyte, which makes strategic decisions for host defense. T cells isolated from the colon or small bowel normally respond very weakly when compared to T cells isolated from blood, while T cells isolated from inflamed intestinal areas in patients with an infection or intestinal cramping and bowel disease due to chronic inflammation respond with still yet another distinct character. This proposal will define those intrinsic properties of the mucosal T cell that enables it to toggle between these responsive and unresponsive states. We recently identified that a chemical balance (called the redox potential) within the T cell regulates whether it can respond weakly or strongly to the outside world. We also showed that by switching the balance within a mucosal T cell to the alternate type this treated intestinal T cell now behaves more like a normal blood-derived T cell. This project will define the exact molecules in the T cell that causes this imbalance. We can then target this molecule for 'correction' and in the future determine whether this type of treatment is an effective therapy for mucosal infections, vaccine development, or mucosal autoimmune-like diseases.